Aging related decreases in NM myosin expression and contractility in a resistance vessel.

Introduction: Vasodilatation in response to NO is a fundamental response of the vasculature, and during aging, the vasculature is characterized by an increase in stiffness and decrease in sensitivity to NO mediated vasodilatation. Vascular tone is regulated by the activation of smooth muscle and nonmuscle (NM) myosin, which are regulated by the activities of myosin light chain kinase (MLCK) and MLC phosphatase. MLC phosphatase is a trimeric enzyme with a catalytic subunit, myosin targeting subunit (MYPT1) and 20 kDa subunit of unknown function. Alternative mRNA splicing produces LZ+/LZ- MYPT1 isoforms and the relative expression of LZ+/LZ- MYPT1 determines the sensitivity to NO mediated vasodilatation. This study tested the hypothesis that aging is associated with changes in LZ+ MYPT1 and NM myosin expression, which alter vascular reactivity. Methods: We determined MYPT1 and NM myosin expression, force and the sensitivity of both endothelial dependent and endothelial independent relaxation in tertiary mesenteric arteries of young (6mo) and elderly (24mo) Fischer344 rats. Results: The data demonstrate that aging is associated with a decrease in both the expression of NM myosin and force, but LZ+ MYPT expression and the sensitivity to both endothelial dependent and independent vasodilatation did not change. Further, smooth muscle cell hypertrophy increases the thickness of the medial layer of smooth muscle with aging. Discussion: The reduction of NM myosin may represent an aging associated compensatory mechanism to normalize the stiffness of resistance vessels in response to the increase in media thickness observed during aging.

Aging and Heart Failure with Preserved Ejection Fraction.

Heart failure is a clinical syndrome characterized by the inability of the cardiovascular system to provide adequate cardiac output at normal filling pressures. This results in a clinical syndrome characterized by dyspnea, edema, and decreased exertional tolerance. Heart failure with preserved ejection fraction (HFpEF) is an increasingly common disease, and the incidence of HFpEF increases with age. There are a variety of factors which contribute to the development of HFpEF, including the presence of hypertension, diabetes, obesity, and other pro-inflammatory states. These comorbid conditions result in changes at the biochemical and cell signaling level which ultimately lead to a disease with a great deal of phenotypic heterogeneity. In general, the physiologic dysfunction of HFpEF is characterized by vascular stiffness, increased cardiac filling pressures, pulmonary hypertension, and impaired volume management. The normal and abnormal processes associated with aging serve as an accelerant in this process, resulting in the hypothesis that HFpEF represents a form of presbycardia. In this article, we aim to review the processes importance of aging in the development of HFpEF by examining the disease and its causes from the biochemical to physiologic level. © 2022 American Physiological Society. Compr Physiol 12: 1-10, 2022.

Loss of Transforming Growth Factor Beta Signaling in Aortic Smooth Muscle Cells Causes Endothelial Dysfunction and Aortic Hypercontractility.

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Marked Up-Regulation of ACE2 in Hearts of Patients With Obstructive Hypertrophic Cardiomyopathy: Implications for SARS-CoV-2-Mediated COVID-19.

OBJECTIVE: To explore the transcriptomic differences between patients with hypertrophic cardiomyopathy (HCM) and controls. PATIENTS AND METHODS: RNA was extracted from cardiac tissue flash frozen at therapeutic surgical septal myectomy for 106 patients with HCM and 39 healthy donor hearts. Expression profiling of 37,846 genes was performed using the Illumina Human HT-12v3 Expression BeadChip. All patients with HCM were genotyped for pathogenic variants causing HCM. Technical validation was performed using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. This study was started on January 1, 1999, and final analysis was completed on April 20, 2020. RESULTS: Overall, 22% of the transcriptome (8443 of 37,846 genes) was expressed differentially between HCM and control tissues. Analysis by genotype revealed that gene expression changes were similar among genotypic subgroups of HCM, with only 4% (1502 of 37,846) to 6% (2336 of 37,846) of the transcriptome exhibiting differential expression between genotypic subgroups. The qRT-PCR confirmed differential expression in 92% (11 of 12 genes) of tested transcripts. Notably, in the context of coronavirus disease 2019 (COVID-19), the transcript for angiotensin I converting enzyme 2 (ACE2), a negative regulator of the angiotensin system, was the single most up-regulated gene in HCM (fold-change, 3.53; q-value =1.30×10-23), which was confirmed by qRT-PCR in triplicate (fold change, 3.78; P=5.22×10-4), and Western blot confirmed greater than 5-fold overexpression of ACE2 protein (fold change, 5.34; P=1.66×10-6). CONCLUSION: More than 20% of the transcriptome is expressed differentially between HCM and control tissues. Importantly, ACE2 was the most up-regulated gene in HCM, indicating perhaps the heart's compensatory effort to mount an antihypertrophic, antifibrotic response. However, given that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses ACE2 for viral entry, this 5-fold increase in ACE2 protein may confer increased risk for COVID-19 manifestations and outcomes in patients with increased ACE2 transcript expression and protein levels in the heart.

The vasculature in HFpEF vs HFrEF: differences in contractile protein expression produce distinct phenotypes.

Both heart failure with reduced (HFrEF) and preserved (HFpEF) ejection fraction are associated with abnormalities of the vasculature, including a resting vasoconstriction and a decrease in sensitivity to nitric oxide (NO) mediated vasodilation. Vascular tone is controlled by the expression and activation of both smooth muscle (SM) and nonmuscle (NM) myosin, and NO mediated vasodilation is regulated by the expression of the leucine zipper positive (LZ+) isoform of the myosin targeting subunit (MYPT1) of myosin light chain phosphatase (MLCP). This study was designed to determine the expression of these contractile proteins in humans with HFrEF and HFpEF vs normal controls. We isolated tertiary mesenteric vessels from remnant biospecimens of patients undergoing partial or total colectomy at Mayo Clinic Rochester from August 2017 to December 2018, and examined the expression of MYPT1 and the LZ + MYPT1 isoform with immunoblots, while 2D SDS-PAGE was used to resolve the phosphorylated and nonphosphorylated regulatory light chains of NM and SM myosin. Our data show that NM myosin expression, as a percentage of total myosin, was 12 ± 3% (controls, n = 6), 7 ± 5% (HFpEF, n = 4) and 37 ± 18% (HFrEF, n = 5, p < 0.05). Total MYPT1 expression was significantly reduced (p < 0.05) in both HFpEF (70 ± 11%) and HFrEF (48 ± 6%); and in HFrEF, LZ + MYPT1 was also depressed (62 ± 19%, <0.05). These results demonstrate that HFrEF and HFpEF are distinct vascular entities, and the changes in protein expression contribute to the vascular abnormalities associated with these diseases. Further in HFpEF, the decrease in MYPT1 would explain why pharmacologic therapies that are designed to activate the NO/cGMP/PKG signaling pathway do not produce a clinical benefit.

Phosphodiesterase expression in the normal and failing heart.

The number of patients with heart failure with reduced ejection fraction (HFrEF) and preserved ejection fraction (HFpEF) is increasing, and for HFpEF, no therapies have clinical benefit. It has been hypothesized that PKG attenuates pathological remodelling, and increasing cGMP would be beneficial for patients with HF. However, neither the RELAX nor NEAT-HFpEF trial showed benefit. But there is still enthusiasm for increasing cGMP in patients with HF, which highlight the need to determine the expression of PDEs in cardiac muscle. This study used immunoblotting to examine the expression of the PDEs that have been suggested to be targets for therapy of HF in both canines (normal and HFpEF) and humans (normal and HFrEF). Our results demonstrate PDE1C and PDE3A are expressed in cardiac muscle, but we could not detect the expression of PDE2A, PDE5A, PDE7A and PDE9A in cardiac tissue lysates from either normal or failing hearts. Thus, one should not expect a clinical benefit for a therapy targeting these PDEs in heart failure, which highlights the importance of rigorous demonstration of the target of therapy prior to undertaking a clinical trial.

HFpEF, a Disease of the Vasculature: A Closer Look at the Other Half.

Patients with heart failure are commonly divided into those with reduced ejection fraction (EF<40%) and those with preserved ejection fraction (HFpEF; EF>50%). For heart failure with reduced EF, a number of therapies have been found to improve patient morbidity and mortality, and treatment is guideline based. However for patients with HFpEF, no treatment has been found to have clinical benefit. To objectively assess treatments for HFpEF, a comprehensive PubMed literature search was performed using the terms HFpEF, heart failure, smooth muscle, myosin, myosin phosphatase, and PKG (up to December 31, 2017), with an unbiased focus on pathophysiology, cell signaling, and therapy. This review provides evidence that could explain the lack of clinical benefit in treating patients with HFpEF with sildenafil and long-acting nitrates. Furthermore, the review highlights the vascular abnormalities present in patients with HFpEF, and these abnormalities of the vasculature could potentially contribute to the pathophysiology of HFpEF. Thus, focusing on HFpEF as a vascular disease could result in the development of novel and effective treatment paradigms.

Regulation of Pulmonary Vascular Smooth Muscle Contractility in Pulmonary Arterial Hypertension: Implications for Therapy.

There are two primary components that produce pulmonary arterial hypertension (PAH); aberrant structural changes (smooth muscle cell proliferation, smooth muscle cell hypertrophy, and the deposition of matrix proteins within the media of pulmonary arterial vessels), and excess vasoconstriction. However, in PAH, the target and aim of all current therapeutic agents is to reduce the contractility of the pulmonary vasculature; prostaglandins, phosphodiesterase inhibitors, guanylate cyclase stimulators, endothelin antagonists, NO inhalation and Rho kinase inhibitors all influence signaling pathways in the pulmonary vascular smooth muscle to decrease vasoconstriction, and hence, pulmonary vascular resistance (PVR). This review will therefore primarily focus on discussing the signaling pathways regulating contractility in pulmonary vascular smooth muscle, the mechanism for current treatments, as well as highlighting potential targets for the development of novel therapies.

MYPT1 isoforms expressed in HEK293T cells are differentially phosphorylated after GTPγS treatment.

Agonist stimulation of smooth muscle is known to activate RhoA/Rho kinase signaling, and Rho kinase phosphorylates the myosin targeting subunit (MYPT1) of myosin light chain (MLC) phosphatase at Thr696 and Thr853, which inhibits the activity of MLC phosphatase to produce a Ca2+ independent increase in MLC phosphorylation and force (Ca2+ sensitization). Alternative mRNA splicing produces four MYPT1 isoforms, which differ by the presence or absence of a central insert (CI) and leucine zipper (LZ). This study was designed to determine if Rho kinase differentially phosphorylates MYPT1 isoforms. In HEK293T cells expressing each of the four MYPT1 isoforms, we could not detect a change in Thr853 MYPT1 phosphorylation following GTPγS treatment. However, there is differential phosphorylation of MYPT1 isoforms at Thr696; GTPγS treatment increases MYPT1 phosphorylation for the CI+LZ- and CI-LZ- MYPT1 isoforms, but not the CI+LZ+ or CI-LZ+ MYPT1 isoforms. These data could suggest that in smooth muscle Rho kinase differentially phosphorylates MYPT1 isoforms.

The emperor's new clothes: PDE5 and the heart.

Phosphodiesterase-5 (PDE5) is highly expressed in the pulmonary vasculature, but its expression in the myocardium is controversial. Cyclic guanosine monophosphate (cGMP) activates protein kinase G (PKG), which has been hypothesized to blunt cardiac hypertrophy and negative remodeling in heart failure. Although PDE5 has been suggested to play a significant role in the breakdown of cGMP in cardiomyocytes and hence PKG regulation in the myocardium, the RELAX trial, which tested effect of PDE5 inhibition on exercise capacity in patients with heart failure with preserved ejection fraction (HFpEF) failed to show a beneficial effect. These results highlight the controversy regarding the role and expression of PDE5 in the healthy and failing heart. This study used one- and two-dimensional electrophoresis and Western blotting to examine PDE5 expression in mouse (before and after trans-aortic constriction), dog (control and HFpEF) as well as human (healthy and failing) heart. We were unable to detect PDE5 in any cardiac tissue lysate, whereas PDE5 was present in the murine and bovine lung samples used as positive controls. These results indicate that if PDE5 is expressed in cardiac tissue, it is present in very low quantities, as PDE5 was not detected in either humans or any model of heart failure examined. Therefore in cardiac muscle, it is unlikely that PDE5 is involved the regulation of cGMP-PKG signaling, and hence PDE5 does not represent a suitable drug target for the treatment of cardiac hypertrophy. These results highlight the importance of rigorous investigation prior to clinical trial design.

Differential phosphorylation of LZ+/LZ- MYPT1 isoforms regulates MLC phosphatase activity.

The vascular response to NO is due, in part, to a Ca(2+) independent activation of myosin light chain (MLC) phosphatase, a trimeric enzyme of 20kDa, 38kDa catalytic and 110-130kDa myosin targeting (MYPT1) subunits. Alternative mRNA splicing produces MYPT1 isoforms that differ by the presence or absence of a central insert (CI) and a leucine zipper (LZ), and the presence of a LZ+ MYPT1 isoform is important for protein kinase G (PKG) mediated activation of MLC phosphatase. This study was designed to determine the molecular basis for the differential sensitivity of the vasculature to NO. Our results demonstrate that the presence of the MYPT1 LZ domain is required for PKG to both phosphorylate MYPT1 at S668 and activate MLC phosphatase. Further for LZ+ MYPT1 isoforms, an S668A MYPT1 mutation prevents the PKG mediated, Ca(2+) independent activation of MLC phosphatase. These data demonstrate that differential PKG mediated S668 phosphorylation of LZ+/LZ- MYPT1 isoforms could be important for determining the diversity in the sensitivity of the vasculature to NO mediated vasodilatation. Thus, the relative expression of LZ+/LZ- MYPT1 isoforms, in part, defines the vascular response to NO and NO based vasodilators, and therefore, plays a role in the regulation of vascular tone in both health and disease.

Cardiac fibrosis in end-stage human heart failure and the cardiac natriuretic peptide guanylyl cyclase system: regulation and therapeutic implications.

Left ventricular assist device (LVAD) support has been used in the treatment of end-stage heart failure (HF), however use of anti-fibrotic co-therapies may improve prognosis. Natriuretic peptides (NPs) possess anti-fibrotic properties through their receptors, GC-A/GC-B/NPR-C. We sought to evaluate cardiac fibrosis and the endogenous NP system in end-stage HF with and without LVAD therapy and to assess the anti-fibrotic actions of the dual GC-A/-B activator CD-NP in vitro. Collagen (Col) protein content was assessed by Picrosirius Red staining and NPs, NP receptors, and Col I mRNA expression were determined by qPCR in LV tissue from patients in end-stage HF (n=13), after LVAD support (n=5) and in normal subjects (n=6). Col I mRNA and protein levels in cardiac fibroblasts (CFs) pretreated with CD-NP were compared to those of BNP or CNP pretreatment. The LV in end-stage HF was characterized by higher Col I mRNA expression and Col protein deposition compared to normal which was sustained after LVAD support. ANP and BNP mRNA expressions were higher while CNP was lower in end-stage HF LV. GC-A expression did not change while GC-B and NPR-C increased compared to normal LV. The changes in NP system expression were not reversed after LVAD support. In vitro, CD-NP reduced Col I production stimulated by TGF-beta 1 greater than BNP or CNP in CFs. We conclude that the failing LV is characterized by increased fibrosis and reduced CNP gene expression. LVAD support did not reverse Col deposition nor restore CNP production, suggesting a therapeutic opportunity for CD-NP.

Anti-remodeling effects of rapamycin in experimental heart failure: dose response and interaction with angiotensin receptor blockade.

While neurohumoral antagonists improve outcomes in heart failure (HF), cardiac remodeling and dysfunction progress and outcomes remain poor. Therapies superior or additive to standard HF therapy are needed. Pharmacologic mTOR inhibition by rapamycin attenuated adverse cardiac remodeling and dysfunction in experimental heart failure (HF). However, these studies used rapamycin doses that produced blood drug levels targeted for primary immunosuppression in human transplantation and therefore the immunosuppressive effects may limit clinical translation. Further, the relative or incremental effect of rapamycin combined with standard HF therapies targeting upstream regulators of cardiac remodeling (neurohumoral antagonists) has not been defined. Our objectives were to determine if anti-remodeling effects of rapamycin were preserved at lower doses and whether rapamycin effects were similar or additive to a standard HF therapy (angiotensin receptor blocker (losartan)). Experimental murine HF was produced by transverse aortic constriction (TAC). At three weeks post-TAC, male mice with established HF were treated with placebo, rapamycin at a dose producing immunosuppressive drug levels (target dose), low dose (50% target dose) rapamycin, losartan or rapamycin + losartan for six weeks. Cardiac structure and function (echocardiography, catheterization, pathology, hypertrophic and fibrotic gene expression profiles) were assessed. Downstream mTOR signaling pathways regulating protein synthesis (S6K1 and S6) and autophagy (LC3B-II) were characterized. TAC-HF mice displayed eccentric hypertrophy, systolic dysfunction and pulmonary congestion. These perturbations were attenuated to a similar degree by oral rapamycin doses achieving target (13.3±2.1 ng/dL) or low (6.7±2.5 ng/dL) blood levels. Rapamycin treatment decreased mTOR mediated regulators of protein synthesis and increased mTOR mediated regulators of autophagy. Losartan monotherapy did not attenuate remodeling, whereas Losartan added to rapamycin provided no incremental benefit over rapamycin alone. These data lend support to investigation of low dose rapamycin as a novel therapy in human HF.

The role of pulmonary vascular contractile protein expression in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is associated with refractory vasoconstriction and impaired NO-mediated vasodilatation of the pulmonary vasculature. Vascular tone is regulated by light chain (LC) phosphorylation of both nonmuscle (NM) and smooth muscle (SM) myosins, which are determined by the activities of MLC kinase and MLC phosphatase. Further, NO mediated vasodilatation requires the expression of a leucine zipper positive (LZ+) isoform of the myosin targeting subunit (MYPT1) of MLC phosphatase. The objective of this study was to define contractile protein expression in the pulmonary arterial vasculature and vascular reactivity in PAH. In severe PAH, compared to controls, relative LZ+MYPT1 expression was decreased (100 ± 14% vs. 60 ± 6%, p<0.05, n=7-8), and NM myosin expression was increased (1 5 ± 4% vs. 53 ± 5% of total myosin, p<0.05, n=4-6). These changes in contractile protein expression should alter vascular reactivity; following activation with Ang II, force activation and relaxation were slowed, and sustained force was increased. Further, the sensitivity to ACh-mediated relaxation was reduced. These results demonstrate that changes in the pulmonary arterial SM contractile protein expression may participate in the molecular mechanism producing both the resting vasoconstriction and the decreased sensitivity to NO-mediated vasodilatation associated with PAH.

Altered reactivity of tertiary mesenteric arteries following acute myocardial ischemia.

BACKGROUND: It is unknown if cardiac ischemia has any deleterious effect on the contractile properties of nonischemic, peripheral vascular beds. Thus, the objective of the present study was to determine whether acute myocardial ischemia results in peripheral vascular dysfunction. METHODS AND RESULTS: This study characterized force maintenance and the sensitivity to acetylcholine (ACh)-mediated smooth muscle (SM) relaxation of tertiary (3rd) mesenteric arteries from Sprague-Dawley rats following 30 min of myocardial ischemia. Both the phosphorylation of nonmuscle (NM) light chain (LC) and SM-LCs as well as the expression of myosin phosphatase targeting subunit 1 (MYPT1) were also determined. Our data demonstrate that acute myocardial ischemia resulted in vascular dysfunction of 3rd mesenteric vessels, characterized by decreases in force maintenance, ACh- and cGMP-mediated SM relaxation, the phosphorylation of NM-LCs and SM-LCs, and MYPT1 expression. Ischemia was also associated with an increase in protein polyubiquitination, suggesting that during ischemia MYPT1 is targeted for degradation or proteolysis. CONCLUSION: Acute myocardial ischemia produces peripheral vascular dysfunction; the changes in LC phosphorylation and MYPT1 expression result in a decrease in both tone and the sensitivity to NO-mediated SM relaxation of the peripheral vasculature.

Sildenafil and B-type natriuretic peptide acutely phosphorylate titin and improve diastolic distensibility in vivo.

BACKGROUND: In vitro studies suggest that phosphorylation of titin reduces myocyte/myofiber stiffness. Titin can be phosphorylated by cGMP-activated protein kinase. Intracellular cGMP production is stimulated by B-type natriuretic peptide (BNP) and degraded by phosphodiesterases, including phosphodiesterase-5A. We hypothesized that a phosphodiesterase-5A inhibitor (sildenafil) alone or in combination with BNP would increase left ventricular diastolic distensibility by phosphorylating titin. METHODS AND RESULTS: Eight elderly dogs with experimental hypertension and 4 young normal dogs underwent measurement of the end-diastolic pressure-volume relationship during caval occlusion at baseline, after sildenafil, and BNP infusion. To assess diastolic distensibility independently of load/extrinsic forces, the end-diastolic volume at a common end-diastolic pressure on the sequential end-diastolic pressure-volume relationships was measured (left ventricular capacitance). In a separate group of dogs (n=7 old hypertensive and 7 young normal), serial full-thickness left ventricular biopsies were harvested from the beating heart during identical infusions to measure myofilament protein phosphorylation. Plasma cGMP increased with sildenafil and further with BNP (7.31±2.37 to 26.9±10.3 to 70.3±8.1 pmol/mL; P<0.001). Left ventricular diastolic capacitance increased with sildenafil and further with BNP (51.4±16.9 to 53.7±16.8 to 60.0±19.4 mL; P<0.001). Changes were similar in old hypertensive and young normal dogs. There were no effects on phosphorylation of troponin I, troponin T, phospholamban, or myosin light chain-1 or -2. Titin phosphorylation increased with sildenafil and BNP, whereas titin-based cardiomyocyte stiffness decreased. CONCLUSION: Short-term cGMP-enhancing treatment with sildenafil and BNP improves left ventricular diastolic distensibility in vivo, in part by phosphorylating titin.

MYPT1 protein isoforms are differentially phosphorylated by protein kinase G.

Smooth muscle relaxation in response to NO signaling is due, in part, to a Ca(2+)-independent activation of myosin light chain (MLC) phosphatase by protein kinase G Iα (PKGIα). MLC phosphatase is a trimeric complex of a 20-kDa subunit, a 38-kDa catalytic subunit, and a 110-133-kDa myosin-targeting subunit (MYPT1). Alternative mRNA splicing produces four MYPT1 isoforms, differing by the presence or absence of a central insert and leucine zipper (LZ). The LZ domain of MYPT1 has been shown to be important for PKGIα-mediated activation of MLC phosphatase activity, and changes in LZ+ MYPT1 isoform expression result in changes in the sensitivity of smooth muscle to NO-mediated relaxation. Furthermore, PKGIα has been demonstrated to phosphorylate Ser-694 of MYPT1, but phosphorylation at this site does not always accompany cGMP-mediated smooth muscle relaxation. This study was designed to determine whether MYPT1 isoforms are differentially phosphorylated by PKGIα. The results demonstrate that purified LZ+ MYPT1 fragments are rapidly phosphorylated by PKGIα at Ser-667 and Ser-694, whereas fragments lacking the LZ domain are poor PKGIα substrates. Mutation of Ser-667 and Ser-694 to Ala and/or Asp showed that Ser-667 phosphorylation is more rapid than Ser-694 phosphorylation, suggesting that Ser-667 may play an important role in the activation of MLC phosphatase. These results demonstrate that MYPT1 isoform expression is important for determining the heterogeneous response of vascular beds to NO and NO-based vasodilators, thereby playing a central role in the regulation of vascular tone in health and disease.

A dynamic approach reveals non-muscle myosin influences the overall smooth muscle cross-bridge cycling rate.

Recent evidence suggests that non-muscle myosin IIB (NMIIB) contributes to smooth muscle contraction. This study was designed to determine the effects of NMIIB on the cross-bridge cycling rate. The cross-bridge cycling rate was investigated using sinusoidal analysis. Frequency analysis revealed two asymptotes in the Bode plot of the data; and the intersection of the asymptotes (corner frequency) was higher for the B(+/-) strain (8.73+/-1.10 Hz vs 16.56+/-1.26 Hz, P<0.05), consistent with a higher overall cross-bridge cycling rate in heterozygous NMIIB KO (B(+/-)) vs WT mice. These results demonstrate that because of their long duty cycle, NMIIB cross-bridges act as an internal load on smooth muscle myosin to decrease the overall cross-bridge cycling rate and muscle V(max) during force maintenance.